The extent that drugs and other substances can penetrate living tissue is clinically important. For example, there is evidence that poor penetration of some anti-cancer drugs limits their effectiveness. The density of blood vessels in cancer tissue can be significantly lower than non-cancerous tissue (1,2). The increased separation of blood vessels in cancer tissue caused by the proliferation of cancer cells leads to a reduction in the ability of molecules supplied by the blood to reach all cells within a tissue (3,4). Inadequate penetration of drugs or other substances may be a problem in other disease states as well, such as infection (e.g. gangrene). Accordingly, systems have been developed in the past for assessing in vitro the penetration potential of anti-cancer agents and the like (5,6).
The most commonly used method for assessing the penetration of agents into living tissue is the multi-cellular spheroid (5). Multi-cellular spheroids are a conglomeration of up to 106 animal cells, which form spheres of tissue and are grown in a flask in which the media is stirred (spinner culture). Penetration of agents into such multi-cellular spheroids can be assessed by applying the agent for a period of time and then either sectioning the spheroids using a cryotome and examining them microscopically or dissociating them and examining individual cells (7,8).
The use of the spheroid system for measuring penetration is effective, but has several significant limitations. The geometry of the penetration into spheroids is almost inverse of some situations encountered in nature, i.e. in many instances drugs diffuse radially outward from capillaries within tissue, while with spheroids the agent diffuses from the outer or maximum surface area toward the center of the sphere.
There are two main drawbacks to using spheroids to measure penetration of an agent. The first is that the concentration of the agent at different depths of penetration cannot always be determined easily. Usually, after a period of time has lapsed to allow penetration of the test agent, the spheroids are removed from the incubating medium and quickly frozen so they may then be sectioned and evaluated. When the agent is fluorescent it can be microscopically evaluated. If radiolabeled (which is expensive and more difficult), a photographic emulsion is applied and then the section is microscopically evaluated. The agent must bind to the cells to permit a detection method involving the dissociation of cells from the spheroid and analysis using flow cytometry. If the agents are not readily identifiable (i.e. colored, fluorescent or radiolabeled) spheroids cannot be used to directly assess their penetration.
The second drawback to using spheroids is that a gradient of energy status, proliferation and other biological parameters forms inwards from the surface of spheroids, which makes it difficult to assess an agent's penetration based on its effect on cells at different depths within the culture. Generally the cells furthest removed from the site of drug exposure are either quiescent or slowly dividing and will therefore have a reduced sensitivity to many drugs (9).
The present invention is an extension of a system for growing multi-layered cell cultures which is the subject of U.S. Pat. No. 5,602,028 issued Feb. 11, 1997 and is hereby incorporated in its entirety by reference. The '028 patent relates to an apparatus and method for growing multi-layered cell cultures on permeable membranes. Each culture is submerged in a liquid growth medium, which contacts both surfaces thereof. The growth medium is circulated past the culture, such as by continuous stirring of the media, to deliver nutrients and oxygen to the culture. Preferably each cell culture is allowed to grow into a multi-layered, substantially planar cell culture mat on its respective membrane. In one embodiment of the invention the membrane and cell culture mat may be transferred to a partition wall between a pair of side by side chambers each containing a liquid medium. The cell cultures may be used for measuring the rate of penetration of an agent, such as a potential drug, through tissue. The drug or other substance to be tested may be inoculated into one of the chambers and the amount or “flux” of drug or other substance passing into the other of the chambers may be measured over time. Such flux measurements may be used to estimate the rate and degree of penetration of the test drug or other substance.
There are several drawbacks to using measurements of flux through multi-layered cell cultures to measure penetration of a drug or other substance. Flux studies are relatively laborious and time-consuming and are not suited to high throughput automated screening. Moreover, flux data is often difficult to interpret. The need has therefore arisen for a system and method for measuring the penetration of a drug or other substance within a multi-layered cell culture using a biological assay. The assay can be used to predict the penetration of the test substance in living tissue.